IiAGL6 participates in the regulation of stamen development and pollen formation in Isatis indigotica.

The AGL6 (AGMOUSE LIKE 6) gene is a member of the SEP subfamily and functions as an E-class floral homeotic gene in the development of floral organs. In this study, we cloned IiAGL6, the orthologous gene of AGL6 in Isatis indigotica. The constitutive expression of IiAGL6 in Arabidopsis thaliana resulted in a late-flowering phenotype and the development of curly leaves during the vegetative growth period. Abnormal changes in floral organ development were observed during the reproductive stage. In woad plants, suppression of IiAGL6 using TRV-VIGS (tobacco rattle virus-mediated virus-induced gene silencing) decreased the number of stamens and led to the formation of aberrant anthers. Similar changes in stamen development were also observed in miRNA-AGL6 transgenic Arabidopsis plants. Yeast two-hybrid and BiFC tests showed that IiAGL6 can interact with other MADS-box proteins in woad; thus, playing a key role in defining the identities of floral organs, particularly during stamen formation. These findings might provide novel insights and help investigate the biological roles of MADS transcription factors in I. indigotica.

IiSVP of Isatis indigotica can reduce the size and repress the development of floral organs.

KEY MESSAGE: IiSVP of Isatis indigotica was cloned and its expression pattern was analyzed. Ectopic expression of IiSVP in Arabidopsis could delay the flowering time and reduce the size of the floral organs. SVP (SHORT VEGETATIVE PHASE) can negatively regulate the flowering time of Arabidopsis. In the present work, the cDNA of IiSVP, an orthologous gene of AtSVP in I. indigotica, was cloned. IiSVP was highly expressed in rosette leaves, inflorescences and petals, but weakly expressed in sepals, pistils and young silicles. The results of subcellular localization showed that IiSVP was localized in nucleus. Bioinformatics analysis indicated that this protein was a MADS-box transcription factor. Constitutive expression of IiSVP in Arabidopsis thaliana resulted in decrease of the number of petals and stamens, and curly sepals were formed. In IiSVP transgenic Arabidopsis plants, obvious phenotypic variations in flowers could be observed, especially the size of the floral organs. In comparison with the wild-type plants, the size of petals, stamens and pistil in IiSVP transgenic Arabidopsis plants was decreased significantly. In some transgenic plants, the petals were wrapped by the sepals. Yeast two-hybrid experiments showed that IiSVP could form higher-order complexes with other MADS proteins, including IiSEP1, IiSEP3, IiAP1 and IiSEP4, but could not interact with IiSEP2. In this work, it was proved that the flowering process and the floral development in Arabidopsis could be affected by IiSVP from I. indigotica Fortune.

Functional identification of the different regions in B-class floral homeotic MADS-box proteins IiAP3 and IiPI from Isatis indigotica.

APETALA3 (AP3) and PISTILLATA (PI) are B-class MADS-box floral homeotic genes of Arabidopsis and are involved in specifying the identity of petals and stamens. In the present work, IiAP3 and IiPI, the respective orthologous genes of AP3 and PI, were cloned from Isatis indigotica. By expressing in ap3-6 and pi-1 homozygous mutant and in wild-type Arabidopsis under the control of AP3 promoter or CaMV 35S promoter, we demonstrated that IiAP3 and IiPI were functionally equivalent to AP3 and PI of Arabidopsis. Referring to previous reports and the research results in the present work, expression patterns of AP3 and PI homologs are not the same in different angiosperms possessing diverse floral structures. It suggests that the alterations in expression may contribute to the changing morphology of flowers. To further determine the relationship between IiAP3 and IiPI, the coding sequences of the different structural regions in these two proteins were swapped with each other, and the data collected from transgenic Arabidopsis plants of the chimeric constructs suggested that MADS domain was irreplaceable for the function of IiAP3, K domain of IiAP3 was involved in specifying the identity of stamens, K domain of IiPI was mainly related to the formation of petals, and C-terminal region of IiPI was involved in characterization of stamens. In addition, a complete KC region of these two proteins was more effective in phenotypic complementation of the mutants.

SEPALLATA--like genes of Isatis indigotica can affect the architecture of the inflorescences and the development of the floral organs.

BACKGROUND: The architecture of inflorescence and the development of floral organs can influence the yield of seeds and have a significant impact on plant propagation. E-class floral homeotic MADS-box genes exhibit important roles in regulation of floral transition and differentiation of floral organs. Woad (Isatis indigotica) possesses unique inflorescence, floral organs and fruit. However, very little research has been carried out to determine the function of MADS-box genes in this medicinal cruciferous plant species. RESULTS: SEPALLATA orthologs in I. indigotica were cloned by degenerate PCR. The sequence possessing the highest identity with SEP2 and SEP4 of Arabidopsis were named as IiSEP2 and IiSEP4, respectively. Constitutive expression of IiSEP2 in Columbia (Col-0) ecotype of Arabidopsis led to early flowering, and the number of the flowers and the lateral branches was reduced, indicating an alteration in architecture of the inflorescences. Moreover, the number of the floral organs was declined, the sepals were turned into carpelloid tissues bearing stigmatic papillae and ovules, and secondary flower could be produced in apetalous terminal flowers. In 35S::IiSEP4-GFP transgenic Arabidopsis plants in Landsberg erecta (Ler) genetic background, the number of the floral organs was decreased, sepals were converted into curly carpelloid structures, accompanied by generation of ovules. Simultaneously, the size of petals, stamens and siliques was diminished. In 35S::IiSEP4-GFP transgenic plants of apetalous ap1 cal double mutant in Ler genetic background, the cauliflower phenotype was attenuated significantly, and the petal formation could be rescued. Occasionally, chimeric organs composed of petaloid and sepaloid tissues, or petaloid and stamineous tissues, were produced in IiSEP4 transgenic plants of apl cal double mutant. It suggested that overexpression of IiSEP4 could restore the capacity in petal differentiation. Silencing of IiSEP4 by Virus-Induced Gene Silencing (VIGS) can delay the flowering time, and reduce the number and size of the floral organs in woad flowers. CONCLUSION: All the results showed that SEPALLATA-like genes could influence the architecture of the inflorescence and the determinacy of the floral meristems, and was also related to development of the floral organs.

Role of melatonin in UV-B signaling pathway and UV-B stress resistance in Arabidopsis thaliana.

Melatonin (N-acetyl-5-methoxytryptamine) plays important roles in plant defences against a variety of biotic and abiotic stresses, including UV-B stress. Molecular mechanisms underlying functions of melatonin in plant UV-B responses are poorly understood. Here, we show that melatonin effect on molecular signalling pathways, physiological changes and UV-B stress resistance in Arabidopsis. Both exogenous and endogenous melatonin affected expression of UV-B signal transduction pathway genes. Experiments using UV-B signalling component mutants cop1-4 and hy5-215 revealed that melatonin not only acts as an antioxidant to promote UV-B stress resistance, but also regulates expression of several key components of UV-B signalling pathway, including ubiquitin-degrading enzyme (COP1), transcription factors (HY5, HYH) and RUP1/2. Our findings indicate that melatonin delays and subsequently enhances expression of COP1, HY5, HYH and RUP1/2, which act as central effectors in UV-B signalling pathway, thus regulating their effects on antioxidant systems to protect the plant from UV-B stress.

Metabolomics analysis identifies metabolites associated with systemic acquired resistance in Arabidopsis.

BACKGROUND: Systemic acquired resistance (SAR) is a type of plant defense response that provides a long-lasting resistance to broad-spectrum pathogens in uninfected distal tissues following an initial localized infection. However, little information is available at present on the biological basis of SAR at the molecular level, especially in uninfected distal leaves. METHODS: In the present work, we used two SAR-inducing pathogens, avirulent Pseudomonas syringae pv. maculicola ES4326 harboring avrRpm1 (Psm avrRpm1) and virulent P. syringae pv. maculicola ES4326 (Psm ES4326), to induce SAR in Arabidopsis ecotype Col-0. A metabolomics approach based on ultra-high-performance liquid chromatography (UPLC) coupled with mass spectrometry (MS) was used to identify SAR-related metabolites in infected local leaves, and in uninfected distal leaves. RESULTS: Differentially accumulated metabolites were distinguished by statistical analyses. The results showed that both the primary metabolism and the secondary metabolism were significantly altered in infected local leaves and in uninfected distal leaves, including phenolic compounds, amino acids, nucleotides, organic acids, and many other metabolites. CONCLUSIONS: The content of amino acids and phenolic compounds increased in uninfected distal leaves, suggesting their contribution to the establishment of SAR. In addition, 2'-hydroxy-4, 4', 6'-trimethoxychalcone, phenylalanine, and p-coumaric acid were identified as potential components which may play important roles both in basic resistance and in SAR. This work provides a reference for understanding of the metabolic mechanism associated with SAR in plants, which will be useful for further investigation of the molecular basis of the systemic immunity.

Tobacco NtabSPL6-2 can enhance local and systemic resistances of Arabidopsis thaliana to bacterial and fungal pathogens.

NtabSPL6-2 of Nicotiana tabacum was introduced into Arabidopsis by Agrobacterium-mediated floral-dip method. Compared to wild-type Col-0 plants, the arrangement of cauline leaves in NtabSPL6-2 transgenic plants was converted into opposite from simple and alternate, and the margin of rosette leaves was serrated. NtabSPL6-2 transgenic plants possessed a significantly greater fresh weight. Subcellular localization by fusion with GFP confirmed that the encoded product of NtabSPL6-2 existed in the nucleus. The leaves of NtabSPL6-2 transgenic plants exhibited an enhanced capacity to restrain the bacterial reproduction after infection by Pseudomonas syringae, accompanied by higher expression of the pathogenesis-related gene PR1 in the infiltrated leaves, indicating NtabSPL6-2 could improve the defense response of Arabidopsis to P. syringae at the local sites. Similarly, it was confirmed that NtabSPL6-2 could enhance the systemic acquired resistance of Arabidopsis in response to P. syringae. In addition, the area of necrotic plaque appearing on the transgenic leaves inoculated with Botrytis cinerea was smaller and accompanied by an upregulation of PR1 and PR5, indicating NtabSPL6-2 transgenic leaves were less susceptible to the fungal pathogen. Moreover, there was less accumulation of reactive oxygen species (H2O2 and O2-) and malondialdehyde in the local infected sites of transgenic plants, whereas the wild-type Col-0 plants were more oxidatively injured after infestation by B. cinerea.

Cloning of a SEPALLATA4-like gene (IiSEP4) in Isatis indigotica Fortune and characterization of its function in Arabidopsis thaliana.

E-class MADS-box genes, SEPALLATA (SEP), participate in various aspects of plant development together with B-, C- and D-class MADS-box genes. IiSEP4, a homologous gene of SEP4, was cloned from Isatis indigotica. IiSEP4 was highly expressed in sepals, and its mRNA was mildly detected in leaves, inflorescences, flowers, stamens and young silicles. Constitutive expression of IiSEP4 in Arabidopsis thaliana caused early flowering, accompanied by the reduction of flowers and floral organs. Moreover, the sepals in some flowers were transformed into carpelloid structures with stigmatic papillae, and obviously accompanied by ovule formation. Yeast two-hybrid assays demonstrated that IiSEP4 interacts with other woad MADS proteins to determine the identity of floral organs. These findings reveal the important roles of IiSEP4 in floral development of I. indigotica. The results of this study can lay a foundation for further study on biological functions of MADS transcriptional factors in I. indigotica.

Ectopic expression of IiSHP2 from Isatis indigotica Fortune, a PLE-lineage MADS-box gene, influences leaf, floral organ and silique morphology in Arabidopsis thaliana.

In order to ascertain the regulatory mechanism of fruit development in Isatis indigotica Fortune, the complementary DNA (cDNA) sequence of the SHATTERPROOF 2 (SHP2) orthologous gene was identified by Rapid Amplification of cDNA Ends technology and the corresponding gene was named IiSHP2. The expression pattern of IiSHP2 was determined by quantitative reverse transcription-polymerase chain reaction and wild-type Col-0 Arabidopsis plants were transformed with the IiSHP2 gene using Agrobacterium tumefaciens and the floral-dip method. Expression analyses indicated that IiSHP2 was highly expressed in flowers, silicles and seeds. Compared to wild-type plants, IiSHP2 transgenic lines bolted earlier. Detailed phenotypic observations showed that the size of the rosette and cauline leaves in transgenic lines was reduced and the cauline leaves of the transgenic lines were incurved and displayed a funnel-like shape. During the reproductive growth stage, IiSHP2 transgenic plants produced shortened sepals and the flower buds were not encapsulated completely. Moreover, the petals of the transgenic lines were converted into stamineous tissues, accompanied by exposed stamens, short malformed siliques and wrinkled valves, indicating a severe decline in fertility. These experimental conclusions are valuable as a reference for the breeding of medicinal plants.

A SEPALLATA1-like gene of Isatis indigotica Fort. regulates flowering time and specifies floral organs.

An orthologous gene of SEPALLATA1, designated as IiSEP1, was isolated from Isatis indigotica. The genomic DNA of IiSEP1 is 3.1 Kb in length. The full-length cDNA of IiSEP1 is 1481 bp and contains a 756 bp ORF encoding a 251-amino-acid protein. Sequence comparison revealed that IiSEP1 belonged to the MADS-box gene family. IiSEP1 contains 7 exons and 6 introns, showing similar exon-intron structure with Arabidopsis SEP1. Phylogenetic analysis suggested that IiSEP1 belonged to AGL2/SEP subfamily and was likely to be an I. indigotica ortholog of Arabidopsis SEP1. Quantitative real-time PCR showed that IiSEP1 was predominantly expressed in the reproductive organs. Ectopic expression of IiSEP1 in Arabidopsis resulted in early flowering, accompanied with the reduction of inflorescence number and the production of terminal flower on the top of the main stems. Moreover, IiSEP1 overexpressing flowers generated numerous variations in phenotype. The sepals were changed into petal-sepal mosaic structures or displayed carpelloid features, and transparent ovules were formed in internal surface of these sepals. In addition, some flowers were constituted by sepals and pistil, but lacked petals and stamens. Taken together, IiSEP1 might play important roles in reproductive growth of I. indigotica and could affect the morphogenesis of flowers and fruits.

Constitutive expression of NtabSPL6-1 in tobacco and Arabidopsis could change the structure of leaves and promote the development of trichomes.

The coding sequence of NtabSPL6-1 was cloned by high-fidelity PCR with specific primers and was used in construction of a binary vector for overexpression. Wild-type Col-0 Arabidopsis plants and Qinyan95 tobacco leaves were transformed using floral dip and leaf disc methods, respectively. Phenotypic observation showed that constitutive expression of NtabSPL6-1 in Arabidopsis could promote the development of trichomes on leaf epidermis and influence the growth pattern of cauline leaves. In tobacco, ectopic expression of NtabSPL6-1 led to dwarfism of the plants and alteration of the leaf structure, accompanied by changes of the glandular trichomes in development. At the same time, the self-regulation capability of NtabSPL6-1 was determined by yeast two-hybrid system. The results indicated that SBP-C terminal domain and C terminal domain of NtabSPL6-1 possessed strong transcriptional activation ability; the intact protein, N terminal domain, and the first peptide fragment in N terminal domain possessed weak transcriptional activation ability; and the second and the third peptide fragments in N terminal domain had no transcriptional activation ability, suggesting the N terminal domain of NtabSPL6-1 could block the activity of the C terminal domain. NtabSPL6-1 may affect the resistance of plants to biotic stress factors indirectly by regulation of the trichome growth.

Ectopic expression of IiFUL isolated from Isatis indigotica could change the reproductive growth of Arabidopsis thaliana.

The coding sequence of IiFUL in Isatis indigotica was isolated and was used in transformation of Arabidopsis. IiFUL overexpressing Arabidopsis plants exhibited early flowering phenotype, accompanied with the reduction of flower number and the production of terminal flower on the top of the main stems. In development process, the flowers located on the top of the main stems generated a lot of variations in phenotype, including abnormal swelling of pistil, withering and numerical change of stamens and petals, appearance of stigmatoid tissues and naked ovules at the margin or inside of sepals. Besides, secondary flower could be formed within the flowers on the top of the main stems. These observations illustrated that IiFUL mainly affected the development of inflorescence meristems and pistils, but its ectopic expression could also disturb the normal growth of other floral organs. Moreover, the fertile siliques produced by the lateral inflorescences of IiFUL overexpressing Arabidopsis plants showed indehiscent phenotype, and the shape of the cauline leaves was changed significantly. The results of quantitative real-time PCR revealed that higher transcriptional levels of IiFUL could be detected in flowers and silicles of I. indigotica. In comprehensive consideration of the previous reports about the dehiscence phenotype of Arabidopsis siliques and the fact that the siliques of IiFUL overexpressing Arabidopsis plants were indehiscent in the present work, it can be speculated that high expression of IiFUL in pericarp is likely the reason why the silicles of I. indigotica possess an indehiscent phenotype.

Metabolomic analysis reveals the relationship between AZI1 and sugar signaling in systemic acquired resistance of Arabidopsis.

The function of AZI1 in systemic acquired resistance of Arabidopsis was confirmed by investigation of the phenotypic features of wild-type Col-0, AZI1 T-DNA knockout and AZI1 overexpressing plants after infection with virulent and avirulent Pseudomonas syringae. Real-time quantitative PCR and Northern blotting analyses showed that the transcript abundances of PR genes increased significantly in local and systemic leaves of wild-type Col-0 and AZI1 overexpressing plants challenged with avirulent P. syringae, whereas the mRNA accumulation of PR genes was obviously attenuated in local and systemic leaves of AZI1 T-DNA knockout plants after localized infiltration with avirulent Psm avrRpm1. The changes of metabolomic profiles in distal leaves of three types of materials infected with avirulent P. syringae were determined by (1)H NMR spectrometry and data mining showed that the soluble carbonhydrates might function as signal substances in the systemic immunity of Arabidopsis. At the same time, the expression of the sugar signaling genes in local and distal leaves after infection of avirulent P. syringae was compared. As a result, it was found that the transcript abundances of sugar signaling genes, including SUS1, SUS2, SUS3, SUS6, SUT1, HXK1, HXK2, SNRK1.2, ERD6, TPS1, TOR, SNRK1.1, SNRK1.3 and bZIP11, were obviously changed in distal leaves of different materials with the modulated AZI1 activities, indicating sugar-related genes are involved in regulation of the systemic immunity mediated by AZI1. These results also illustrated that the immune system associated with sugar molecules probably was an important part of the systemic acquired resistance in Arabidopsis.

Characterization and phylogenetic analysis of fifteen NtabSPL genes in Nicotiana tabacum L. cv. Qinyan95.

Fifteen SPL (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) genes were identified and characterized in Nicotiana tabacum L. cv. Qinyan95. The exon-intron structures of these genes were determined according to the coding sequences confirmed by RT-PCR and the genomic DNA sequences downloaded from the databases in Sol Genomics Network, and thirteen of them were found to carry the response element of miR156. To elucidate the origin of the validated NtabSPL genes, multiple alignments of the nucleotide sequences encompassing the open reading frames were conducted by using the orthologs in N. tabacum, Nicotiana sylvestris, Nicotiana tomentosiformis, and Nicotiana otophora. The results showed that six NtabSPL genes were derived from a progenitor of N. sylvestris, and nine NtabSPL genes were derived from a progenitor of N. tomentosiformis, further corroborating that N. tabacum came from the interspecific hybridization between the ancestors of N. sylvestris and N. tomentosiformis. In contrast to previous statements about highly repetitive sequences, the genome of N. tabacum mainly retained the paternal-derived SPL genes in diploidization process. Phylogenetic analyses based on the highly conserved SBP (SQUAMOSA PROMOTER BINDING PROTEIN) domains and the full-length amino acid sequences reveal that the SPL proteins of tobacco, tomato, and Arabidopsis can be categorized into eight groups. It is worth noting that N. tabacum contains seven NtabSPL6 genes originated from two parental genomes and NtabSPL6-2 possesses a GC-AG intron. In addition, transgenic tobacco plants harboring Arabidopsis Pri-miR156A were generated by Agrobacterium-mediated transformation method, and the constitutive expression of miR156 could obviously inhibit the activity of the NtabSPL genes containing its target site, suggesting the function of miR156 is conservative in tobacco and Arabidopsis.

Plasmalemma localisation of DOUBLE HYBRID PROLINE-RICH PROTEIN 1 and its function in systemic acquired resistance of Arabidopsis thaliana.

The protein encoded by AtDHyPRP1 (DOUBLE HYBRID PROLINE-RICH PROTEIN 1) contains two tandem PRD-8CMs (proline-rich domain-eight cysteine motif) and represents a new type of HyPRPs (hybrid proline-rich proteins). Confocal microscopy to transgenic Arabidopsis plants revealed that AtDHyPRP1-GFP was localised to plasmalemma, especially plasmodesmata. AtDHyPRP1 mainly expressed in leaf tissues and could be induced by salicylic acid, methyl jasmonate, virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and avirulent P. syringae pv. tomato DC3000 harbouring avrRPM1 (Pst avrRPM1), suggesting it is involved in defence response of Arabidopsis thaliana (L. Heynh.). After treatments with bacterial suspension of virulent Pst DC3000 or conidial suspension of Botrytis cinerea, AtDHyPRP1 overexpressing lines exhibited enhanced resistance, whereas AtDHyPRP1 RNA interference lines became more susceptible to the pathogens with obvious chlorosis or necrosis phenotypes. In systemic acquired resistance (SAR) analyses, distal leaves were challenged with virulent Pst DC3000 after inoculation of the primary leaves with avirulent Pst avrRPM1 (AV) or MgSO4 (MV). Compared with MV, the infection symptoms in systemic leaves of wild-type plants and AtDHyPRP1 overexpressing lines were significantly alleviated in AV treatment, whereas the systemic leaves of AtDHyPRP1 RNAi lines were vulnerable to Pst DC3000, indicating AtDHyPRP1 was functionally associated with SAR.

Expression of recombinant EARLI1, a hybrid proline-rich protein of Arabidopsis, in Escherichia coli and its inhibition effect to the growth of fungal pathogens and Saccharomyces cerevisiae.

EARLI1 is an Arabidopsis gene with pleiotropic effects previously shown to have auxiliary functions in protecting plants against freezing-induced cellular damage and promoting germinability under low-temperature and salinity stresses. Here we determined whether recombinant EARLI1 protein has anti-fungal activity. Recombinant EARLI1 protein lacking its signal peptide was produced in Escherichia coli BL21(DE3) using isopropyl ß-d-1-thiogalactopyranoside (IPTG) induction and the prokaryotic expression vector pET28a. Expression of EARLI1 was analyzed by Western blotting and the protein was purified using affinity chromatography. Recombinant EARLI1 protein was applied to fungal cultures of Saccharomyces cerevisiae, Botrytis cinerea and Fusarium oxysporum, and membrane permeability was determined using SYTOX green. Full-length EARLI1 was expressed in S. cerevisiae from the GAL1 promoter using 2% galactose and yeast cell viability was compared to control cells. Our results indicated that application of recombinant EARLI1 protein to B. cinerea and F. oxysporum could inhibit the growth of the necrotrophic fungi. Besides, addition of the recombinant protein to liquid cultures of S. cerevisiae significantly suppressed yeast growth and cell viability by increasing membrane permeability, and in vivo expression of the secreted form of EARLI1 in S. cerevisiae also had a remarkable inhibition effect on the growth of yeast cells.

The HyPRP gene EARLI1 has an auxiliary role for germinability and early seedling development under low temperature and salt stress conditions in Arabidopsis thaliana.

The effect of the hybrid proline-rich protein (HyPRP) gene EARLI1 on the rate of germination (germinability) of Arabidopsis seeds and seedling growth under low temperature and salt stress conditions was investigated. EARLI1 was induced during germination in embryonic tissues, and was strongly expressed in certain parts of young seedlings. Comparisons of control, overexpressing (OX), and knockout (KO) lines indicated that higher than wild type levels of EARLI1 improved germinability, root elongation, and reduction of sodium accumulation in leaves under salt stress, as well as germinability under low-temperature stress. Abscisic acid (ABA) contents were relatively low after prolonged salt stress, suggesting that EARLI1 has an ABA-independent effect on germinability under these conditions. Overexpression of EARLI1 during germination enhanced the sensitivity of seeds to exogenously applied ABA, suggesting that EARLI1 has an ABA-dependent negative effect on seed germinability under high ABA stress conditions. Well-known stress response marker genes such as COR15a, KIN1, P5SC1, and RD29 were unaffected whereas P5SC2, RD22, or RAB18 were only slightly affected in OX and KO plants. The pleiotropic effects of EARLI1 during stress and an absence of strong regulatory effects on stress marker genes suggest that this HyPRP gene has an auxiliary role for various stress protection responses in Arabidopsis.

Cold-inducible expression of AZI1 and its function in improvement of freezing tolerance of Arabidopsis thaliana and Saccharomyces cerevisiae.

AZI1 (AZELAIC ACID INDUCED 1) of Arabidopsis thaliana could be induced by azelaic acid and was involved in priming of systemic plant immunity. In the present work, expression of AZI1 in response to low temperature was investigated via RNA gel blot analysis. AZI1 could be induced slowly by cold stress and more than 6h treatment at 4°C was required to detect an increase in mRNA abundance. However, the high expression state could not be maintained stably and would decline to basal level when the plants were transferred to room temperature. In order to clarify the function of AZI1 in resistance to abiotic stresses, overexpressing, RNA interference and T-DNA knockout lines of this gene were used in electrolyte leakage assays. Overexpression of AZI1 resulted in reduced electrolyte leakage during freezing damage. In contrast, AZI1 knockdown and knockout lines showed increased tendencies in cellular damage after freezing treatment. To further validate the potential resistance of AZI1 to low-temperature stress, Saccharomyces cerevisiae cells were transformed with pESC-AZI1 in which AZI1 was under the control of GAL1 promoter. Compared to yeast cells containing empty pESC-URA, the survival rate of yeast cells harboring AZI1 increased obviously after freezing treatment. All these results suggested that AZI1 might be multifunctional and associated with cold tolerance of Arabidopsis.

Multicellular genesis of leaf primordium was demonstrated via chimaeric transgenic plant of maize (Zea mays L.) regenerated from Type II calli.

Type-II embryonic calli were induced from immature embryos of maize (Zea mays L.) genotype YD and bombarded with beta-glucuronidase gene. Bombarded calli were proliferated on normal N6 medium for 2 weeks at 26°C in the dark and selected on N6 medium containing 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 mg/l phosphinothricin (PPT) but without casamino acids and proline under the same conditions for 14 days. Regeneration was carried out on hormone-free MS medium containing 5 mg/l phosphinothricin at 26°C under 3000 lux illumination. Plants over 8 cm were transplanted into soil and sprayed with 250 mg/l phosphinothricin when two new leaves appeared. Except normal transgenic plants, chimaeric transgenics also were regenerated in the present work. The expression pattern of beta-glucuronidase gene in leaves of chimaeric transgenic plant revealed that more than one cell formed leaf primordium at the initial stage, and filial cells stemed from each cell in leaf primordium arranged in a row longitudinally from leaf base to leaf apex. There was a clear boundary as a straight line between the area formed by transformed cells and the area formed by normal cells. A hypothesis was put forward that the primitive cells in leaf primordium divided in a longitudinal style, resulted in leaf elongation, then the filial cells divided transversally and synchronously toward the outside to broaden the leaf.

Changes of gentiopicroside synthesis during somatic embryogenesis in Gentiana macrophylla.

IN VITRO plant regeneration of Gentiana macrophylla Pall. and determination of gentiopicroside content during somatic embryogenesis are described in the present work. The highest percentage of embryogenic callus formation was observed in Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L 6-benzylaminopurine (BA). Calli were subcultured on MS medium containing 1.0 mg/L 2,4-D, 1.0 mg/L BA and 500 mg/L lactalbumin hydrolysate (LH) at intervals of 25 days. A higher frequency of somatic embryo maturation was achieved on MS medium containing B5 vitamins (MB) supplemented with different concentrations of 1-naphthaleneacetic acid (NAA) and BA than with a combination of NAA and kinetin (KT). Addition of AgNO(3) improved maturation of somatic embryos while thidiazuron (TDZ) promoted vitrification. The gentiopicroside contents of embryogenic calli and globular-, heart-, torpedo-, and cotyledon-shaped embryoids were determined by high-performance liquid chromatography (HPLC). Gentiopicroside was not detectable in embryogenic calli, but in all types of somatic embryos. The highest gentiopicroside content was observed in cotyledon-shaped embryoids, reaching more than 12 mg/g dry weight.